Current treatment of chronic hepatitis C based on combination of peginterferon and ribavirin is only effective in about half of the patients and is accompanied by substantial side effects. Developing new classes of drugs against HCV is crucial. Phosphorothioate oligonucleotides (PS-ONs) have a sequence-independent antiviral activity against HIV-1 by inhibiting virus-cell fusion. Because viral entry is a highly conserved mechanism, this antiviral action of PS-ONs may be effective against infection by other enveloped viruses with type I or II fusion mechanisms. By applying the cell culture system described above, we assessed whether PS-ONs inhibit HCV infection and to evaluate the antiviral mechanism of action of PS-ONs. Various forms of PS-ONs and the control phosphodiester oligonucleotides (PO-ONs) were synthesized and evaluated in infectious HCV cell culture system systems. To test the efficacy of PS-ON in vivo, human hepatocytes transplanted uPASCID mice were inoculated with infectious HCV and treated with the PS-ON. The PS-ONs exhibited potent inhibitory activities in both cell culture-generated HCV-JFH1 (HCVcc) and HCV pseudo-particles (HCVpp) systems. This inhibitory activity was size and phosphorothioation dependent but sequence independent. The control PO-ONs had no inhibitory activity against HCV infection. The PS-ONs had no effect on viral replication in the HCV replicon system and binding of HCV-LPs to cells, indicating that the target of inhibition by PS-ONs is at the post-binding, cell-entry step. In human hepatocyte-engrafted uPASCID mice, the PS-ONs also appeared to efficiently block de novo HCV infection. The PS-ONs (amphipathic DNA polymers) are a promising new class of antiviral compounds that inhibit HCV fusion and entry. HCV dependencies on the host machinery are both intricate and extensive. Each of these host dependencies is a potential therapeutic target. Previous efforts have been successful in discovering important steps in HCV replication, yet many fundamental processes in the viral lifecycle remain uncharacterized. Using RNAi-based genetics and an infectious HCV cell culture system, we performed an unbiased genome-wide screen to identify host factors required for productive HCV infection. We applied a two-part screening protocol to identify host factors involved in the complete viral lifecycle, from viral entry to production of infectious virus. A validation screen was subsequently performed to minimize potential off-target effects. 512 genes were identified in the initial screen and 262 were confirmed by the validation assay. We identified 238 host susceptibility factors (HSFs) and 24 host resistance factors (HRFs), the majority of which were not previously linked to HCV. Of these 262 validated hits, 45 target late-stage viral infection. Integrative bioinformatics analyses of these host genes and other published database revealed a broad and complex dependency of HCV on cellular processes and molecular functions, and also implicated novel cellular signaling pathways modulating HCV infection. Several key pathways including TGF-beta, ErbB, MAPK, focal adhesion and ubiquitin proteolysis are particularly enriched in the bioinformatics analysis. By applying various virologic assays and molecular techniques, a comprehensive map of cellular pathways and machineries that are associated with each steps of HCV lifecycle, including viral entry, intracellular trafficking, viral RNA replication and translation, polyprotein processing, virion assembly and secretion, are being established. A global identification and characterization of HCV-host interactions will significantly advance our understanding of HCV-related pathogenesis, and hence illuminates potentially valuable targets for prophylactic and therapeutic interventions. Based on the infectious HCV cell culture system, we are also setting up a cell-based assay for high-throughput screening (HTS) of small molecule chemical library in collaboration with the NIH Chemical Genomics Center. The NCGC has a large collection of over 250,000 chemical compounds and has established the facility for HTS. By performing cell-based HTS, we hope to identify novel targets and lead compounds for HCV therapeutic development. The identification of the hepatitis C virus (HCV) strain JFH-1 enabled the successful development of infectious cell culture systems. Although this strain replicates efficiently and produces infectious virus in cell culture, the replication capacity and pathogenesis in vivo are still undefined. To assess the in vivo phenotype of the JFH-1 virus, cell culture-generated JFH-1 virus (JFH-1cc) and patient serum from which JFH-1 was isolated were inoculated into chimpanzees. Both animals became HCV RNA-positive 3 days after inoculation, but showed low-level viremia and no evidence of hepatitis. HCV viremia persisted 8 and 34 weeks in JFH-1cc and patient serum-infected chimpanzees, respectively. Immunological analysis revealed that HCV-specific immune responses were similarly induced in both animals. Sequencing of HCV at various time points of infection revealed more substitutions in the patient serum-inoculated chimpanzee and the higher level of sequence variations seemed to be associated with a prolonged infection in this animal. A common mutation G838R in the NS2 region emerged early in both chimpanzees. This mutation enhances viral assembly leading to an increase in viral production in transfected or infected cells. Our study shows that the HCV JFH-1 strain causes attenuated infection and low pathogenicity in chimpanzees, and is capable of adapting in vivo with a unique mutation conferring enhanced replicative phenotype.